Bone-mimetic porous hydroxyapatite/whitlockite scaffolds: preparation, characterization and interactions with human mesenchymal stem cells

Bone-mimetic highly porous Mg-substituted calcium phosphate scaffolds, composed of hydroxyapatite (HAP) and whitlockite (WH), were synthesized by hydrothermal method at 200 °C, using calcium carbonate skeletons of cuttlefish bone, ammonium dihydrogenphosphate (NH 4 H 2 PO 4 ) and magnesium chloride hexahydrate (MgCl 2 × 6H 2 O) or magnesium perchlorate (Mg(ClO 4 ) 2 ) as reagents. The effect of Mg content on the compositional and morphological properties of scaffolds was studied by means of X-ray diffraction, Fourier transform infrared spectroscopy, thermogravimetric analysis and scanning electron microscopy (SEM) with energy-dispersive X-ray analysis. Structural refinements performed by Rietveld method indicated that Mg 2+ ions were preferentially incorporated into the WH phase. SEM images of all prepared scaffolds showed that the interconnected structure of the cuttlefish bone was completely maintained after the hydrothermal synthesis. Results of compression tests showed a positive impact of the whitlockite phase on the mechanical properties of scaffolds. Human mesenchymal stem cells (hMSCs) were cultured on scaffolds in osteogenic medium for 21 days. Immunohistochemical staining showed that Mg-CaP scaffolds with the HAP:WH wt ratio of 90:10 and 70:30 exhibited higher expression of collagen type I and osteocalcin than pure HAP scaffold. Calcium deposition was confirmed by Alizarin Red staining. Positive effect of Mg 2+ ions on the differentiation of hMSCs on porous 3D scaffolds was also confirmed by reverse transcription-quantitative polymerase chain reaction analysis. Graphical abstract